This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Improved understanding of the structural basis of electrostatic effects in proteins is necessary for correlation of structure with function. Most previous studies have focused on surface ionizable residues. Their general properties and their contributions to stability and function are relatively well understood. One of the remaining problems in protein electrostatics concerns the properties of internal ionizable groups. The pKa values of these groups are governed by dielectric properties of proteins that are poorly understood. To examine this issue we engineered a family of proteins with ionizable groups (Lys, Arg, Asp, Glu) at 25 internal positions. A hyperstable variant of staphylococcal nuclease was used for these purposes. 98 out of 100 variants that were made fold into a native-like state. The pKa values of these internal residues have been measured. The majority of the pKa values are shifted in the direction that promotes the neutral state (elevated for acidics and depressed for basic residues), some by nearly 6 pKa units. These shifts suggest that the dehydration experienced by the ionizable groups in their buried positions is not fully compensated for by contacts with polar or ionizable groups.